DESCRIPTION: Numerous chemical agents react with DNA to form exocyclic modified DNA bases which promote replication errors and mutations, if unrepaired. The long-term goal of this project focuses on identifying human enzymes that remove cyclic adducts from DNA and defining their mechanism of action. The experimental approach utilizes a defined site-specific adduct-containing DNA substrate to probe for and purify DNA-glycosylases from human cell-free extracts. Structurally related adducts incorporated into defined oligonucleotides include 1,N6-etheno A (EA), 3-N4-EC, 1,N2-EG, N2,3-EG, 1,N6-benzetheno A (pBQ-A), 3, N4-pBQ-C, 1,N2, pBQ-G and 1,N6-ethano-A. These adducts are formed after DNA treatment with chloroacetaldehyde and p-benzoquinone. Using cell-free extracts from HeLa or human placenta, novel glycosylases will be identified following cleavage induction and polyacrylamide gel electrophoresis or HPLC detection of the modified exocyclic base. DNA glycosylases that act on these substrates will be purified, characterized, microsequenced and compared in the protein data base to other proteins. The objective is to extend our understanding of enzyme-substrate recognition. Four specific aims are listed that direct this project. 1) To construct site-specific oligonucleotides containing various nucleotides modified by bifunctional agents. The detailed synthetic chemistry, phosphoramidite, and oligoneucleotide synthesis will be conducted under other NIH grants (CA47723 and ES07363). 2) To test for excision of these modified bases in human cell-free extracts. Band-shift, binding kinetics and footprinting experiment will be conducted to describe the enzyme-substrate interaction. 3) To investigate the range of substrate specificity and sequence context effects of repair rates for EC-DNA glycosylase, benzethenoA-DNA glycosylase and benzetheno-C-DNA glycosylase. 4) To purify to homogeneity recently identified or other new human glycosylase, characterize the activities and obtain protein micro-sequencing information.